*3.2. Replacement of L. donovani SUMO*

To test the importance of *SUMO* for *L. donovani* viability and/or proliferation, we targeted *SUMO* for CRISPR/Cas9-mediated replacement, following an established protocol [34,39]. The 5′ sgRNAand 3′ sgRNA-coding sequences, along with the upstream and downstream flanking primers were designed as shown in Figure 2A, together with two primer pairs to test for the presence of *SUMO*. The selection marker gene cassettes from plasmids pTPURO and pTBLAST were amplified using the

upstream and downstream flanking primers to yield 1.9 kb PCR products (Figure 2B). Those, together with the 5′ - and 3′ -sgRNA-coding oligonucleotides (Figure 2C) were transfected into *L. donovani* expressing both the Cas9 recombinase and the T7 RNA polymerase (*L. donovani* (Cas9/T7-RNAP)). The transfectants were then selected under IC<sup>95</sup> (95%-inhibiting concentration) for puromycin and blasticidin. Selected parasites were cloned by limiting dilution [42] and tested for the presence of *SUMO* by PCR with two independent primer pairs. Figure 2D shows that all tested clones remained positive for *SUMO*.

The success of CRISPR-mediated gene replacement is very dependent on a perfect match between gene sequences and the annealing sgRNA regions. We therefore tested whether the sgRNA pair was able to basepair with the *SUMO* coding sequence. For this, we repeated the transfection of sgRNA-coding oligonucleotides and selection marker cassettes in an *L. donovani* strain over expressing *SUMO* from episomal gene copies to create SUMO–/–/+ parasites. In five out of six clones, we could verify the loss of the chromosomal *SUMO* alleles. This confirms the specificity of the sgRNAs and selection marker cassette amplificates. We conclude that replacement of *SUMO* is only possible in the presence of ectopic SUMO gene copies, giving strong evidence for an essential role of *SUMO* in viability and/or proliferative capacity of *L. donovani*.

As C-terminal processing by SENP/Ulp2 is thought to be critical for conjugation and polymerization of SUMO, but also for de-SUMOylation, we next targeted the putative SENP ortholog for replacement.

**Figure 2.** Replacement of *SUMO* in *Leishmania donovani.* (**A**) Schematic representation of LdBPK\_080480.1 replacement using the CRISPR/Cas9 technology. *SUMO*-targeting sgRNAs (grey) and the replacement cassettes were PCR-amplified and transfected into a Cas9/T7-RNAP-expressing *L. donovani* strain. Two sets of genotyping primers were used to test for the presence of the gene of interest (GOI) (**B**) Gene-specific replacement cassettes amplified from pTPURO or pTBLAST vector were analyzed by agarose gel electrophoresis and ethidium bromide staining. The position of the DNA size marker is indicated on the left, the primers used are indicated on the right. (**C**) Amplified sgRNA-coding sequences were separated on a 1% agarose gel and stained with ethidium bromide. (**D**) Genotyping of putative gene replacement mutant clones with primer pairs 7+8 or 5+6. PCR products were analyzed by 1% agarose gel electrophoresis. Positions of DNA size markers are shown to the left; the primer pairs are indicated on the right. (**E**) Genotyping of gene replacement mutants in the SUMO over expression background (SUMO−/−/+) indicated primer pairs.
